This proposal examines the role of intracellular and extracellular calcium in the event which immediately follow platelet stimulation by agonists, but precede the platelet release reaction and aggregation. Two general approaches will be taken. The first half of the project examines the mechanisms that maintain the free Ca++ concentration in the cytosol of unstimulated platelets below 10-6 M and modulate the increase in cytosol Ca++ concentration that occurs after stimulation. Included in these studies will be the continued development of a new method for separating the internal membrane systems of platelet, including the dense tubular system, from the platelet's plasma membrane. This will permit key mechanisms for calcium transport, such as Ca-Mg-ATPase, to be localized to specific membrane systems and allow examination of the role of the dense tubular system as a labile site for internal Ca++ sequestration and release. In the second part of the project, our recent observation that ADP and epinephrine can increase the uptake of 45Ca++ by intact platelets will be extended to distinguish 45CA++ binding to the platelet surface from 45Ca++ entry into the cell. Since changes in Ca++ binding to the platelet surface may reflect conformational changes in membrane components (with concommitant exposure of new binding sites) or movement of Ca++ to the inner surface, the number and varying affinities of calcium binding sites on unstimulated platelets will be measured in order to allow the effects of agonists to be determined. The mechanism for agonist-related 45Ca++ entry into the platelet will be examined using 45CA++ efflux to study both bidirectional passive changes in membrane permeability and unidirectional active transport. Metallochromic indicators and a calcium electrode will be used to distinguish net calcium entry from 45Ca++ exchange. Finally, platelet membranes will be enriched with cholesterol to examine the relationship between altered Ca++ uptake and the previously observed increase in platelet sensitivity to agonists that occurs with cholesterol loading.